cancers Review RLIP76: A Structural and Functional Triumvirate Jasmine Cornish, Darerca Owen and Helen R. Mott * Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK; [email protected] (J.C.); [email protected] (D.O.) * Correspondence: [email protected] Simple Summary: The RLIP76 protein is present at high levels in multiple cancers, compared with levels in normal cells. In cancer cells it is thought to be important for removal of chemotherapeutics, while in normal cells it has been implicated in endocytosis, stress response and mitochondrial fission. Although RLIP76 is a potential target for ovary, breast, lung, colon, prostate and kidney cancers, only the middle third of the protein has been structurally characterized. Understanding the full structure of RLIP76 will help us to better understand the signalling pathways in which it is involved and by extension its role in cancer. Abstract: RLIP76/RalBP1 is an ATP-dependent transporter of glutathione conjugates, which is overexpressed in various human cancers, but its diverse functions in normal cells, which include endocytosis, stress response and mitochondrial dynamics, are still not fully understood. The protein can be divided into three distinct regions, each with its own structural properties. At the centre of the protein are two well-defined domains, a GTPase activating protein domain targeting Rho family small G proteins and a small coiled-coil that binds to the Ras family small GTPases RalA and RalB. In engaging with Rho and Ral proteins, RLIP76 bridges these two distinct G protein families. The N-terminal region is predicted to be disordered and is rich in basic amino acids, which may Citation: Cornish, J.; Owen, D.; Mott, mediate membrane association, consistent with its role in transport. RLIP76 is an ATP-dependent H.R. RLIP76: A Structural and transporter with ATP-binding sites within the N-terminus and the Ral binding domain. Furthermore, Functional Triumvirate. Cancers 2021, RLIP76 is subject to extensive phosphorylation, particularly in the N-terminal region. In contrast, 13, 2206. https://doi.org/10.3390/ the C-terminal region is thought to form an extensive coiled-coil that could mediate dimerization. cancers13092206 Here, we review the structural features of RLIP76, including experimental data and computational predictions, and discuss the implications of its various post-translational modifications. Academic Editors: Gabriella D’Orazi and Sanjay Awasthi Keywords: RalBP1; RLIP76; small G protein; RhoGAP; Ral Received: 14 March 2021 Accepted: 29 April 2021 Published: 4 May 2021 1. Introduction RLIP76, also known as RalBP1, is a 76 kDa protein that was simultaneously discovered Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in by three groups as a downstream effector of the Ral family of small GTPases. It was shown published maps and institutional affil- that RLIP76 binds to both RalA and RalB, but not to other closely related proteins within iations. the Ras branch of the Ras superfamily of small GTPases [1–3]. Small G proteins cycle between a GDP bound, inactive state and a GTP bound, active state. They can slowly hydrolyse GTP, but for efficient hydrolysis a GTPase activating protein (GAP) is required to stimulate their intrinsic GTPase activity. A second set of proteins, the guanine nucleotide exchange factors (GEFs) stimulate the exchange of the bound GDP for GTP. The Ral proteins Copyright: © 2021 by the authors. function downstream of Ras: Ras activates RalGEFs, which convert the Rals to their active, Licensee MDPI, Basel, Switzerland. GTP-bound form. Ras proteins are mutated in approximately 19% of all cancers, with This article is an open access article distributed under the terms and high incidences found in pancreatic (88%) and gastrointestinal cancers (colon, 50%; rectal, conditions of the Creative Commons 50%; small intestine, 26%) [4], highlighting them as an important therapeutic target. Ras Attribution (CC BY) license (https:// therapeutics however, have proven difficult to develop, although some progress is being creativecommons.org/licenses/by/ made with covalent inhibitors against specific mutants (reviewed in [5]), and attention has 4.0/). turned to inhibiting downstream pathways, including the Ral pathway. The Ral proteins Cancers 2021, 13, 2206. https://doi.org/10.3390/cancers13092206 https://www.mdpi.com/journal/cancers Cancers 2021, 13, x 2 of 15 Cancers 2021, 13, 2206 2 of 15 and attention has turned to inhibiting downstream pathways, including the Ral pathway. The Ral proteinsare drivers are drivers of cell growth,of cell growth, survival survival and metastasis and metastasis [6]. Downstream [6]. Downstream of Ral, RLIP76 of itself is Ral, RLIP76overexpressed itself is overexpressed in multiple in tumour multiple types tumour and mediatestypes and resistance mediates to resistance chemotherapeutics to and chemotherapeuticsapoptosis. and It apoptosis. is itself a potentialIt is itself a target potential in ovary, target breast, in ovary, lung, breast, colon, lung, prostate colon, and kidney prostate andcancers kidney (reviewed cancers (reviewed in [7]). in [7]). The RLIP76The protein RLIP76 can proteinbe divided can beinto divided three distinct into three regions, distinct each regions, of which each has of its which has its own interactingown partners. interacting Thus, partners. it can Thus,be thought it can of be as thought a protein of as of athree protein parts, of threewhich parts, may which may act separatelyact or separately synergistically or synergistically to perform tothe perform various thefunctions various that functions have been that ascribed have been ascribed to RLIP76. to RLIP76. 2. Domain Architecture2. Domain Architecture of RLIP76 of RLIP76 RLIP76 comprisesRLIP76 655 comprises residues 655 but residues contains but only contains two recognizable only two recognizable domains in domainsthe in the centre of thecentre protein of the(Figure protein 1). (FigureThe RhoGAP1). The homology RhoGAP homologydomain encompasses domain encompasses residues residues 192–368 [1,8]192–368 and has [1 been,8] and shown has to been stimulate shown the to GTPase stimulate activity the GTPase of the Rho activity family of small the Rho family GTPases Rac1small and GTPases Cdc42 in Rac1 vitro and [1– Cdc423], albeitin very vitro weakly.[1–3], albeit The Ral very binding weakly. domain The Ral (RBD) binding domain (RBD) was identified in a region adjacent to the RhoGAP domain and interacts with both was identified in a region adjacent to the RhoGAP domain and interacts with both RalA RalA and RalB [2,9]. This was subsequently shown to form a short coiled-coil spanning and RalB [2,9]. This was subsequently shown to form a short coiled-coil spanning residues residues 393 and 446, which is sufficient to bind tightly to the Ral proteins [9]. RLIP76 393 and 446, which is sufficient to bind tightly to the Ral proteins [9]. RLIP76 is not closely is not closely related to any other proteins, except within the RhoGAP domain, which is related to any other proteins, except within the RhoGAP domain, which is present in a present in a large number of proteins with diverse domain arrangements [10]. The RhoGAP large number of proteins with diverse domain arrangements [10]. The RhoGAP domain domain and Ral binding domain of RLIP76 are conserved within metazoans, whereas the and Ral binding domain of RLIP76 are conserved within metazoans, whereas the N-ter- N-terminal and C-terminal thirds are only conserved in Danio rerio through to Homo sapiens minal and C-terminal thirds are only conserved in Danio rerio through to Homo sapiens (Figure1B )[11,12]. Ral binding is clearly an essential aspect of its function, since RLIP76 is (Figure 1B) [11,12]. Ral binding is clearly an essential aspect of its function, since RLIP76 only found in organisms that also have Ral [11–13]. is only found in organisms that also have Ral [11–13]. Figure 1. ArchitectureFigure and1. Architecture conservation and of theconservation domains inof RLIP76. the domains (A) Domain in RLIP76. architecture (A) Domain of RLIP76. architecture The central of RhoGAP and Ral bindingRLIP76. domains The are central flanked RhoGAP by structurally and Ral uninvestigated binding domains N- andare flanked C-termini. by Thestructurally C-terminus uninvestigated is predicted to form a coiled coil, whileN- and the C N--termini. terminus The is C predicted-terminus tois bepredicted disordered. to form The a positionscoiled coil, of while two ATPthe N binding- terminus sites is arepre- indicated by inverted triangles.dicted to Known be disordered. interactions The positions are labelled, of two with ATP their binding approximate sites are indicated binding regions by inverted indicated triangles. by arrows. Known interactions are labelled, with their approximate binding regions indicated by arrows. (B) (B) Phylogenetic tree of RLIP76 in a subset of organisms. The schematic (styled as in Figure1A) shows the conserved Phylogenetic tree of RLIP76 in a subset of organisms. The schematic (styled as in Figure 1A) shows features for each organism. Only the RhoGAP and RBD are conserved in Caenorhabditis elegans. The predicted coiled-coil is present in Danio rerio through to Homo sapiens, while the two ATP binding motifs are present in Rattus norvegicus, Mus musculus and Homo sapiens. Cancers 2021, 13, 2206 3 of 15 The N-terminal third of RLIP76 (residues 1–190) binds to several other proteins. It interacts with the interdomain linker of the mu2 subunit of the clathrin adapter complex, AP2, leading to receptor mediated endocytosis of EGFR [14]. Residues 1–180 interact with the Arf GEF, ARNO, in turn activating Arf6, leading to cell spreading and migration [15,16]. Mutation of Ser29RLIP76 and Ser30RLIP76 to alanine inhibits ARNO binding [15]. A region in the N-terminal third of RLIP76 that extends 16 residues into the RhoGAP domain (1–208) has been shown to interact with the ENTH domain of the Epsin family of endocytic adap- tors. Knockdown of Epsin has also been shown to decrease levels of active Arf6 and Rac1, since Epsin inhibits RLIP76 GAP activity [17].